Method for modulating metabolism

ABSTRACT

A method for modulating metabolism is provided which includes the step of providing a consumable composition including an extract containing a compound of Formula I to a subject in need thereof thereby modulating the subject&#39;s metabolism and addressing the underlying pathogenesis of metabolic disorders, such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis and type II diabetes mellitus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/501,230 filed Oct. 14, 2021, which is a continuation of U.S.application Ser. No. 16/961,035, filed on Jul. 9, 2020, issued Nov. 16,2021 as U.S. Pat. No. 11,173,136, which was the National Phase ofInternational Application No. PCT/US2019/012993, filed on Jan. 10, 2019and published on Jul. 18, 2019, as WO 2019/140052, which claims thebenefit of priority of U.S. Provisional Application No. 62/615,671 filedJan. 10, 2018, the content of which is incorporated herein by referencein its entirety.

BACKGROUND

The “Western Diet” has been associated with a global rise in metabolicdisorders such as obesity, type II diabetes mellitus (T2DM), metabolicsyndrome, nonalcoholic fatty liver disease (NAFLD), heart disease, andstroke. Interactions between genetic and environmental factors such asdiet and lifestyle, particularly over-nutrition and sedentary behavior,promote the progression and pathogenesis of these polygenic diet-relateddiseases. Their current prevalence is increasing dramatically toepidemic proportions. Nutrition is probably the most importantenvironmental factor that modulates expression of genes involved inmetabolic pathways and the variety of phenotypes associated withobesity, the metabolic syndrome, and type II diabetes mellitus.Furthermore, the health effects of nutrients may be modulated by geneticvariants.

A 70% ethyl alcohol extract of Tribulus terrestris has been suggested toprovide a protective effect in a model of type I diabetes mellitus(i.e., streptozotocin-induced diabetic rats) by inhibiting oxidativestress (Amin, et al. (2006) Ann. NY Acad. Sci. 1084:391-401).

U.S. Pat. Nos. 8,481,593 and 9,089,499 disclose para-coumaric acidderivatives such as N-trans-feruloyltyramine in topical and cosmeticcompositions for use in inhibiting human tyrosinase and in the treatmentof hyperpigmentation.

An acetone extract from Smilax aristolochiifolia root, which is enrichedfor N-trans-feruloyltyramine, has been suggested to be useful incounteracting some symptoms (e.g., hypertriglyceridemia, insulinresistance, blood pressure, and inflammation) in an injury modelassociated with metabolic syndrome (Amaro, et al. (2014) Molecules19:11366-84).

US 2008/0132544 suggests the use of isolated N-trans-feruloyl tyraminefrom Piper nigrum in a composition for the treatment of visceral fatobesity, T2DM, insulin resistant syndrome and metabolic syndrome.

SUMMARY OF THE INVENTION

The present invention provides a method for modulating metabolism byproviding to a subject in need thereof a consumable composition composedof at least one carrier and an effective amount of an extract comprisinga compound of Formula I, or an isomer, heterodimer, or conjugatethereof:

wherein

-   -   R¹ is present or absent, and when present is a substituent on        one or more ring atoms and is for each ring atom independently a        hydroxy group, halo group, substituted or unsubstituted lower        alkyl group, or substituted or unsubstituted lower alkoxy group;        and    -   the dashed bond is present or absent.

In some embodiments, the compound has the structure of Formula II:

wherein, R² is present or absent, and when present is a hydroxy ormethoxy group; R³ is present or absent, and when present is a hydroxygroup; and R⁴ is present or absent, and when present is a hydroxy ormethoxy group.

Preferably, the extract is an ethanol extract of a member of the genusAllium, Amoracia, Chenopodium, Fagopyrum, Annona, Piper, Eragrostis,Zea, Cannabis, Ipomea, Capsicum, Lycium, Solanum, or Tribulus. In someembodiments, the consumable composition is formulated as a dietarysupplement, food ingredient or additive, a medical food, nutraceuticalor pharmaceutical composition. Ideally, an effective amount of thecomposition provides an improvement in HNF4α activity, insulin-likegrowth factor levels, blood sugar levels, insulin levels, HbA1C levels,C peptide levels, triglyceride levels, free fatty acid levels, blooduric acid levels, microalbuminuria levels, glucose transporterexpression, adiponectin levels, total serum cholesterol levels, highdensity lipoprotein levels, low density lipoprotein levels or acombination thereof. In certain embodiments, the subject has or is atrisk of developing a metabolic disorder, e.g., insulin resistance,hyperglycemia, type II diabetes mellitus, obesity, glucose intolerance,dyslipidemia, hypercholesterolemia, hyperlipoproteinemia,hyperinsulinemia, atherosclerotic disease, coronary artery disease,metabolic syndrome, or hypertension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a dose-response analysis of N-trans-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyltyramine in an assay measuringinsulin promoter activity. Dimethylsulfoxide (DMSO) and alverine (20 μM)were used as negative and positive controls, respectively.

FIG. 2 shows the effect of N-trans-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyltyramine on insulin mRNA levelsas determined by quantitative PCR. DMSO and alverine (20 μM) were usedas negative and positive controls, respectively.

FIG. 3 shows the effect of N-trans-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyltyramine on HNF4α mRNA levels asdetermined by quantitative PCR. DMSO and alverine (20 μM) were used asnegative and positive controls, respectively.

FIG. 4 shows that N-trans-caffeoyltyramine-mediated increases in insulinexpression are inhibited by BI-6015, a known HNF4α antagonist.

FIG. 5 shows the effect of N-trans-caffeoyltyramine andN-trans-feruloyltyramine on estrogenic activity. Assays were carried outin the presence (1 μM) or absence (0 μM) Tamoxifen (Tam) using Alverineand 7005 (CAS No. 380336-90-3) (known HNF4α transcriptional activators)as positive controls and cis-feruloyltyramine and DMSO as negativecontrols.

FIG. 6 demonstrates that N-trans-caffeoyltyramine andN-trans-feruloyltyramine can reverse fat accumulation. T6PNE cells werepretreated for 1 day with 0.06 mM, 0.12 mM or 0.25 mM palmitate at whichtime 15 μM N-trans-caffeoyltyramine or control (DMSO) was added. Cellswere harvested on day 3, 6 and 8 and subjected to staining with Nile Redand Oil Red O. Results are expressed as fold change in Nile Redstaining: +palmitate/10% FBS medium (no palmitate)

FIG. 7 shows that N-trans-caffeoyltyramine increases nuclear expressionof HNF4α in the liver.

FIG. 8 shows that lipid droplet size in the liver is reduced bytreatment with N-trans-caffeoyltyramine.

FIG. 9 shows levels of blood analytes alkaline phosphatase (ALP),alanine transaminase including (ALT), γ-glutamyltransferase (GGT),bilirubin, artresia, total albumin, albumin, blood urea nitrogen (urea),and cholesterol in mice treated with N-trans-caffeoyltyramine or control(DMSO).

FIG. 10 shows triglyceride levels in the liver of mice fed a high fatdiet and treated with N-trans-caffeoyltyramine or control (DMSO).

FIG. 11 shows the effect of N-trans-caffeoyltyramine on HNF4α expressionin the pancreas of mice fed a high fat diet as compared to control(DMSO).

FIG. 12 shows the effect of N-trans-caffeoyltyramine on HNF4α expressionin the intestine of mice fed a high fat diet as compared to control(DMSO).

FIG. 13 shows the amounts of N-trans-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyl tyramine present in ethanolextracts (% of extract, w/w) from a variety of sources includingTribulus terrestris seed (1), Cannabis (hemp) seed hull (2), Annona spp.(atemoya) seed (3), Annona muricata (Guanabana) seed (4), A. cherimola(Cherimoya) leaf (5), Zea mays stalk (6), Tribulus terrestris (GoatHead) seed (7), A. cherimola hardwood (bark and core) (8), Solanumlycopersicum ground pomace (9), S. tuberosum (yellow potato) peel (10),Piper nigrum (black peppercorn) fruit (11), S. tuberosum (purple potato)peel (12), S. tuberosum (red potato) peel (13), S. lycopersicum pomace(14), S. lycopersicum extruded pomace (15), A. muricata (Guanabana)leaves (16), Allium sativum (garlic) bulb (17), S. tuberosum (purplepotato) peel (18), A. montana (Mountain soursop) leaves (19), Z. maysleaves (20), S. tuberosum (purple potato) sprouts (21), A. cherimola(Cherimoya) seed (22), Allium fistulosum (green onion) whole plant (23),S. tuberosum (white potato) peel (24), A. cherimola (Cherimoya)greenwood (25), Cannabis (hemp) leaves (26), S. tuberosum (white potato)peel (27), S. lycopersicum seed (28), S. lycopersicum (Beefsteak) wholefruit (29), A. muricata (Guarabana) skin of unripe fruit (30), A.muricata (Guanabana) ripe fresh fruit (31), A. squamosa (sweetsop) wholefruit (32), Capsicum annuum (serrano pepper) fruit (33), S. tuberosum(Russet potato) peel (34), Lycium barbarum (goji/wolf berry) fruit (35),S. tuberosum (purple potato) core (36), Chenopodium quinoa (quinoa) seed(37), Ipomoea batatas (sweet potato) whole potato (38), Ipomoea batatas(sweet potato) peel (39), Armoracia rusticana (horseradish) root (40),S. tuberosum (Colorado potato) peel (41), Fagopyrum esculentum(buckwheat) hulls (42), Capsicum frutescens (piri piri pepper) fruit(43), S. tuberosum (purple potato) core (44), C. annuum (Thai chili)stems and leaves (45), A. muricata (Guanabana) unripe fruit flesh (46),S. tuberosum (yellow potato) core (47), and Eragrostis tef (teff) seed(48).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides tyramine containing hydroxycinnamic acid amides,which modulate metabolism, in particular HNF4α activity, therebymitigating the adverse effects of free fatty acids in both liver cellsand pancreatic β-cells. The tyramine containing hydroxycinnamic acidamide of this invention are analogs of lead compounds identified intraditional screening assays for agents that modulate known signalingpathways. The tyramine containing hydroxycinnamic acid amides exhibitdose-response HNF4α activity, as initially determined in a T6PNEengineered pancreatic cell, and upregulate insulin gene expression.Further, these compounds show strong, lipid clearing activity in ahepatocyte (hepG2) lipid challenge model of fatty liver disease. Whilenot wishing to be bound by theory, it is believed that the tyraminecontaining hydroxycinnamic acid amides of this invention, modulate HNF4αactivity as a result of higher affinity for the HNF4α binding site thanthe natural ligand, palmitic acid, which down regulates HNF4α activity.Genetic, functional genomic, transcriptomic and clinical evidenceindicate that HNF4α agonists can improve overall metabolic health byenabling the body to maintain sugar and lipid homeostasis. Accordingly,the compounds herein are of use in methods of promoting and/orrecovering healthy HNF4α function, mitigating the adverse effects offree fatty acids, modulating metabolism, and addressing the underlyingpathogenesis of metabolic disorders, such as NAFLD, nonalcoholicsteatohepatitis (NASH) and T2DM. Using the composition of thisinvention, health and well-being are improved and promoted.

Active Compound

This invention provides plant-derived aromatic or more acidic hydroxylgroups arenes, and their use in modulating metabolites attached to withone aromatic metabolism. In one embodiment, the plant-derived aromaticmetabolite is a structural analog of compound 1:

In particular, the invention encompasses a tyramine containinghydroxycinnamic acid amide having the structure of Formula I, or anisomer, salt, homodimer, heterodimer, or conjugate thereof:

wherein

-   -   R¹ is present or absent, and when present is a substituent on        one or more ring atoms (e.g., position 2, 3, and/or 4) and is        for each ring atom independently a hydroxy group, halo group,        substituted or unsubstituted lower alkyl group, or substituted        or unsubstituted lower alkoxy group; and    -   the dashed bond is present or absent.

For the groups herein, the following parenthetical subscripts furtherdefine the groups as follows: “(C_(n))” defines the exact number (n) ofcarbon atoms in the group. For example, “C₁-C₆-alkyl” designates thosealkyl groups having from 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6,or any range derivable therein (e.g., 3-6 carbon atoms)).

The term “lower alkyl” is intended to mean a branched or unbranchedsaturated monovalent hydrocarbon radical containing 1 to 6 carbon atoms(i.e., C₁-C₆-alkyl), such as methyl, ethyl, propyl, isopropyl,tert-butyl, butyl, n-hexyl and the like.

Similarly, a lower alkoxy group is a C₁-C₆-alkoxy group having thestructure —OR wherein R is “alkyl” as defined further above. Particularalkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, iso-butoxy, sec-butoxy, n-pentoxy,1,2-dimethylbutoxy, and the like.

The term “halo” is used herein to refer to chloro (Cl), fluoro (F),bromo (Br) and iodo (I) groups. In particular embodiments, the halogroup is a fluoro group.

In any of the groups described herein, an available hydrogen may bereplaced with an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl,alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy,alkoxyalkoxy, acyl, halo, nitro, cyano, carboxy, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, alkyl sulfonyl, aryl sulfonyl,heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio,heteroaralkylthio, cycloalkyl, or heterocyclyl.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

In some embodiments, R¹ is present and preferably represents independentsubstituents at the para and meta positions. In particular embodiments,R¹ is present and represents a hydroxy group at the para position and ahydroxy or lower alkoxy group at the meta position. In certainembodiments, the tyramine containing hydroxycinnamic acid amide havingthe structure of Formula I is in the trans configuration.

In particular embodiments, the tyramine containing hydroxycinnamic acidamide has a structure of Formula II:

wherein,

-   -   R² is present or absent, and when present is a hydroxy or        methoxy group;    -   R³ is present or absent, and when present is a hydroxy group;        and    -   R⁴ is present or absent, and when present is a hydroxy or        methoxy group.

“Isomer” refers to especially optical isomers (for example essentiallypure enantiomers, essentially pure diastereomers, and mixtures thereof)as well as conformation isomers (i.e., isomers that differ only in theirangles of at least one chemical bond), position isomers (particularlytautomers), and geometric isomers (e.g., cis-trans isomers).

In certain embodiments, the tyramine containing hydroxycinnamic acidamide is one of the following compounds:

A salt of a compound of this disclosure refers to a compound thatpossesses the desired pharmacological activity of the parent compoundand includes: (1) an acid addition salt, formed with an inorganic acidsuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with an organic acid such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2) asalt formed when an acidic proton present in the parent compound isreplaced.

As is known in the art, a homodimer is a molecule composed of twoidentical tyramine containing hydroxycinnamic acid amide subunits. Bycomparison, a heterodimer is a molecule composed of two differenttyramine containing hydroxycinnamic acid amide subunits. Examples ofhomodimers of this invention include but are not limited to across-linked N-trans-feruloyltyramine dimer, a cross-linkedN-trans-caffeoyltyramine dimer and a cross-linked p-coumaroyltyraminedimer. See, for example, King & Calhoun (2005) Phytochemistry66(20):2468-73, which teaches the isolation of a cross-linkedN-trans-feruloyltyramine dimer from potato common scab lesions.Conjugates of monomers of tyramine containing hydroxycinnamic acid amideand other compounds, such as lignan amides. Examples of conjugatesinclude, but are not limited to cannabisin A, cannabisin B, cannabisinC, cannabisin D, cannabisin E and cannabisin F.

Sources of Active Compound

A compound of this invention can be obtained from any suitable botanicalspecies and/or botanical raw material known to possess a compound ofFormula I. Preferably, the compound is provided as an extract comprisingthe compound or a substantially pure compound.

An “extract” refers to a composition containing a compound of Formula I,which is separated from other unwanted substances present in the naturalsource material from which the extract was obtained. In someembodiments, the natural source material is a plant. Plant extracts canbe obtained from any plant tissue including a whole plant; a plant partsuch as shoot vegetative organs/structures (for example, leaves, stemsand tubers), roots, flowers and floral organs/structures. (for example,bracts, sepals, petals, stamens, carpels, anthers and ovules), a seed(including embryo, endosperm, and seed coat) or fruit (the matureovary); a plant tissue (for example, vascular tissue, ground tissue, andthe like); cells (for example, guard cells, egg cells, and the like), orexudates as well as progeny and cultures or cell lines of the same.Preferably, the extract contains compounds that will be found to begenerally recognized as safe (GRAS) for human consumption. Accordingly,in certain embodiments the extract is from an edible source. In thisrespect, the extract is an edible extract.

Extracts can be prepared by freezing, grinding, macerating, pulverizing,fermenting, percolation, decoction, solvent extraction (e.g.,partitioning) or precipitation, treatment with activated charcoal,evaporation, filtration, and/or chromatographic fractionation of thesource material of interest. In this respect, an “extract” of theinvention can be crude, fractionated, sub-fractionated, separated,isolated, enriched or purified, without being limited thereto. The term“crude” means compounds or molecules that have not been entirelyseparated from the components of the original composition in which itwas present. In embodiments pertaining to fractions or sub-fractions, amolecule in crude extract may be subjected to partial separation toprovide a less crude extract containing other substances. In someembodiments, the compound is isolated. The term “isolated” means that acompound or molecule is substantially enriched or purified with respectto the complex cellular milieu in which it naturally occurs, such as ina crude extract. When an isolated molecule is enriched or purified, theabsolute level of purity is not critical and those skilled in the artcan readily determine appropriate levels of purity according to the useto which the material is to be put. In some circumstances, the isolatedmolecule forms part of a composition (for example a more or less crudeextract containing many other substances), which may for example containother components. In other circumstances, the isolated molecule may bepurified to essential homogeneity, for example as determinedspectrophotometrically, by NMR or by chromatography (for example LC-MS).

Suitable solvents for preparing an extract include, e.g., n-pentane,hexane, butane, chloroform, dichloromethane, di-ethyl ether,acetonitrile, water, butanol, isopropanol, ethanol, methanol, glacialacetic acid, acetone, norflurane (HFA134a), ethyl acetate, dimethylsulfoxide, heptafluoropropane (HFA227), and subcritical or supercriticalfluids such as liquid carbon dioxide and water, or a combination thereofin any proportion. When solvents such as those listed above are used,the resultant extract typically contains non-specific lipid-solublematerial. This can be removed by a variety of processes including“winterization”, which involves chilling to a specified temperature,typically −20° C. followed by filtration or centrifugation to removewaxy ballast, extraction with subcritical or supercritical carbondioxide or non-polar sol vents (e.g., hexane) and by distillation.

Extracts enriched for a compound of the invention are ideally obtainedby chromatographic fractionation. Chromatographic fractionationtypically includes column chromatography and may be based on molecularsizing, charge, solubility and/or polarity. Depending on the type ofchromatographic method, column chromatography can be carried out withmatrix materials composed of, for example, dextran, agarose,polyacrylamide, silica, C18, C8, polyvinylpyrrolidone, polystyrene,celite, and phenyl-hexy and can include solvents such as dimethylsulfoxide, pyridine, water, dimethylformamide, methanol, saline,ethylene dichloride, chloroform, propanol, ethanol, isobutanol,formamide, methylene dichloride, butanol, acetonitrile, isopropanol,tetrahydrofuran, dioxane, chloroform/dichloromethane, methanol, hexane,and ethyl acetate.

Typically, the product of the chromatographic step is collected inmultiple fractions, which may then be tested for the presence of thedesired compound using any suitable analytical technique (e.g., thinlayer chromatography, mass spectrometry, and ultraviolet absorption).Fractions enriched in the desired compound may then be selected forfurther purification.

As an alternative, or in conjunction with chromatography,crystallization may be performed to obtain high purity tyraminecontaining hydroxycinnamic acid amides. The solubility of the tyraminecontaining hydroxycinnamic acid amide is adjusted by changingtemperature and/or the composition of the solution, for instance byremoving ethanol, and/or adjusting the pH to facilitate precipitation,followed by filtration or centrifugation of the precipitated crystals oroils. Other suitable methods include, but are not limited to,liquid-liquid extraction, centrifugal partition chromatography oradsorption onto a resin or removal of impurities with resin.

A “substantially pure” preparation of a compound is defined as apreparation having a chromatographic purity (of the desired compound) ofgreater than 95%, more preferably greater than 96%, more preferablygreater than 97%, more preferably greater than 98%, more preferablygreater than 99% and most preferably greater than 99.5%, as determinedby area normalization of an HPLC profile.

The term “extract comprising a compound” encompasses preparations havingat least 2%, preferably greater than 5%, more preferably greater than10% chromatographic purity for the desired compound. Such an extractwill generally contain a greater proportion of impurities, non-targetmaterials and other molecules than a “substantially pure” preparation.

In particular embodiments, an “extract comprising a compound” is a“botanical” product or substance. In this context, “botanical” refers to“products that include plant materials, algae, macroscopic fungi andcombinations thereof” Botanicals are defined by the process steps usedto prepare the extract (e.g., by pulverization, decoction, expression,aqueous and/or ethanol extraction) and provide a quantified amount ofone or more of the compounds of interest.

Ideally, a compound of this invention is extracted and/or purified froma plant. Exemplary plants sources include, but are not limited to,plants in the genera, family, order, genus, species listed in Table 1.

TABLE 1 Order Family Genus Common name Asparagales Amaryllidaceae AlliumGarlic Onion Leek Brassicales Brassicaceae Amoracia HorseradishCaryophyllales Amaranthaceae Chenopodium Quinoa CaryophyllalesPolyconaceae Fagopyrum Buckwheat Manoliales Annonaceae Annona CherimoyaAtemoya Soursop Sweetsop Custard apple Guanabana Piperales PiperaceaePiper Black pepper Poales Poaceae Eragrostis Teff Zea Corn RosalesCannabaceae Cannabis Hemp Solanales Convolvulaveae Ipomea Sweet potatoSolanaceae Capsicum Serrano pepper Thai Chili Piri piri pepper LyceumGoji/wolf berry Solanum Tomato Potato Zygophyllaceae ZygophyllalesTribulus Goat thorn Puncture vine

By way of illustration, an extract containing N-trans-caffeoyltyramineis obtained by grinding or pulverizing the dried fruit of Tribulusterrestris, subjecting the pulverized material to 80% ethanol at roomtemperature, filtering and concentrating the 80% ethanol extract,resuspending the concentrated extract in water, partitioning the aqueoussolution with hexane, adding chloroform to the aqueous layer, andsubjecting the chloroform layer to liquid chromatography with silicagel. See, e.g., Ko, et al. (2015) Internatl. J. Mol. Med. 36(4):1042-8.

An extract containing a tyramine containing hydroxycinnamic acid amidecan be standardized using conventional techniques such ashigh-performance liquid chromatography (HPLC) or high-performancethin-layer chromatography (HPTLC) The term “standardized extract” refersto an extract which is standardized by identifying characteristicingredient(s) or bioactive marker(s) present in the extract.Characterization can be, for example, by analysis of the spectral datasuch as mass spectrum (MS), infrared (IR), ultraviolet (UV) and nuclearmagnetic resonance (NMR) spectroscopic data.Biological Activity

Biological activity of compounds and/or extracts can be determined usingone or more of the well-known biological in vitro assays, in vivo assaysand animal models described in more detail below. Each of these assayswould provide a measure of the activity of the compounds of the presentinvention to provide beneficial effects on cellular endpoints linked tometabolic disorders including but not limited to obesity, T2DM, heartdisease, stroke, fatty liver disease (NAFLD) and nonalcoholicsteatohepatitis (NASH).

Triglyceride Assay in Cultured Hepatocytes.

For measuring triglyceride synthesis in cultured primary hepatocytes,freshly isolated hepatocytes (e.g., from rats) are cultured for 24 hourswith normal media (Dulbecco's modified Eagle Medium (DMEM) with 0.25%bovine serum albumin) in the presence or absence of monounsaturatedand/or saturated fatty acids (e.g., palmitate (C16:0) or oleate (C18:1),or a 2:1 mixture of the two) and presence or absence of an extract orcompound of the invention. Quantitative estimation of hepatictriglyceride accumulation is performed by extraction of hepatic lipidsfrom cell homogenates using chloroform/methanol (2:1) and enzymaticassay of triglyceride mass using an ENZYCHROM™ Triglyceride Assay Kit(Bioassay Systems, Hayward, Calif.).

Adipocyte Glucose Consumption Assay.

Equal amounts (5×10⁵ cells) of 3T3-L1 pre-adipocytes are seeded andcultured in normal D-glucose, DMEM with 10% fetal bovine serum (FBS),penicillin-streptomycin in a humidified atmosphere of 95% air and 5% CO₂at 37° C. When the cells reach 100% confluence, 3T3-L1 pre-adipocytesare induced to be differentiate by treating the culture with 450 mg/dLD-glucose, 0.32 μM insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 1 μMdexamethasone for 2 days. Subsequently, the culture medium of thedifferentiated adipocytes is changed to DMEM containing 450 mg/dLD-glucose with or without the administration of a compound or extract ofthe invention. After 24 hours, the glucose consumption activity isdetermined by measuring the medium glucose concentration with insulinused as the positive control. Protocols and assays for glucose uptakeinto cells are available commercially (e.g., ABCAM: Cambridge, Mass.;Promega: Madison, Wis.).

Insulin Secretory Activity.

Insulin-secreting cells, e.g., rat RIN-m5F cells, are plated into96-well plates and used at subconfluence after a 24-hour incubation.Cells are exposed to 100 μl of sub-toxic concentrations of a compound orextract of the invention and incubated at 37° C. with 5% CO₂ for 3hours. Following treatment, plates are centrifuged at 1000 g for 10minutes and insulin concentration of supernatants is determined using asolid phase two-site enzyme immunoassay, e.g., DRG Ultrasensitive RatInsulin ELISA kit (DRG International, Inc.).

Insulin Promoter Activity.

T6PNE cells (Kiselyuk, et al. (2012) Chem Biol. 19(7):806-818; Kiselyuk,et al. (2010) J. Biomol. Screen 15(6):663-70) are seeded at 2000 cellsper well in 384-well tissue culture plates in the presence of 1 μMtamoxifen and 0.03 mM palmitate. After a 24-hour incubation, a compoundor extract of the invention is added to the cells. Forty-eight hoursafter compound or extract addition, cells are fixed in 4%paraformaldehyde and stained with DAPI. Blue (DAPI) and green (humaninsulin promoter driving GFP) channels are imaged.

Triglyceride Assay in Liver.

Mice are provided a compound or extract of the invention. Liver extractsare prepared by homogenization in 0.25% sucrose with 1 mmol/L EDTA, andlipids are extracted using chloroform/methanol (2:1 v/v) and suspendedwith 5% fatty acid-free bovine serum albumin. Triglyceride levels aremeasured using triglyceride assay reagents (Sigma Chemical Co.).

Hepatic Triglyceride Secretion In Vivo.

This assay employs the use of TRITON WR1339, which inhibits alllipoprotein lipases and therefore clearance of triglycerides from theblood (Millar et al. 2005. J. Lipid Res. 46:2023-2028) Mice are provideda compound or extract of the invention. Subsequently, the mice areinjected with 10% TRITON WR1339 per animal by intravenous (IV) injectionand blood is collected to assess triglycerides at 0 minutes, 1 hour and2 hours. Plasma is separated and assayed for triglycerides. Triglyceridesecretion rates are expressed as milligram per kilogram per hour afternormalizing with their liver weight.

De Novo Lipogenesis Assay.

De novo lipogenesis is thought to be involved in the pathogenesis ofNAFLD (Sanders and Griffin. 2016. Biol. Rev. Camb. Philos. Soc.91(2):452-468). Primary hepatocytes from animals treated with a compoundor extract of the invention are cultured overnight with 10% DMEMcontaining insulin (100 nM) and dexamethasone (1 μM). Cells aresubsequently incubated with 74 KBq/ml (2-14° C.) sodium acetate (2.07GBq/mmol) for 1 hour. The cells are lysed with 1 N NaOH, acidified, andlipids are extracted with petroleum ether. Radioactivity is measured byliquid scintillation counter.

Animal Models of T2DM.

Models of T2DM include but are not limited to leptin-deficient mouse(ob/ob; Orel, et al. (2006) Diabetes 55(12):3335-43; Wang, et al. (2014)Curr. Diabetes Rev. 10(2):131-145), the leptin-receptor-deficient mouse(db/db; Wang, et al. (2014) Curr. Diabetes Rev. 10(2):131-145), theobese Zucker rat (fa/fa; Shiota & Printz (2012) Methods Mol. Biol.933:103-23), the Wistar Kyoto rat (fa/fa; Figlewicz, et al. (1986)Peptides 7:61-65), proopiomelanocortin-deficient mice (POMC^(−/−);Yaswen, et al. (1999) Nat. Med. 5:1066-1070), melanocortin 3 and 4receptor knockout animals (Huszar, et al. (1997) Cell 88:131-141;Butler, et al. (2000) Endocrinology 141(9):3518-21; Mul, et al. (2011)Obesity (Silver Spring) 20(3):612-21; Chen, et al. (2000) Nat. Genet.26(1):97-102), animals overexpressing glucose transporter subtype 4(Shepard, et al. (1993) J. Biol. Chem. 268:22243-22246) andneuron-specific insulin receptor knockout mice (NIRKO mice; Bruning, etal. (2000) Science 289:2122-2125). Reviews of the use of such animalmodels are available (e.g., Chatzigeorgiou et al. 2009. A.J.K. 2012. Br.J. Pharmacol. In Vivo 28:345-358; King, 166:877-894). These models arecharacterized by insulin resistance, hyperglycemia, andhyperinsulinemia, symptoms mirrored in human T2DM. Animals are providedwith a compound or extract of the invention and maximum tolerated doseand improvements in metabolism are evaluated.

Animal Model of Lipodystrophy.

The complete lack of fat tissue (lipodystrophy) leads to similarmetabolic changes as severe obesity and is associated with insulinresistance. Genetically modified mice with a lack of adipose tissue arecharacterized by hyperphagia, hepatic steatosis, hypertriglyceridaemia,insulin resistance and T2DM (Savage (2009) Dis. Model Mech. 2(11-12):554-62). Due to the lack of functional adipose tissue, thesemice are leptin deficient and are of use in assessing the effect of thecompound or extract of this invention on dysregulated metabolism. Suchmodels are useful for demonstrating in vivo response for compounds ofthe present invention and exploring key concepts such as dose-response.

Rat Models of Diet-Induced Obesity.

Outbred Sprague-Dawley rats have been used as a polygenic model ofobesity (Levin, et al. (1997) Am. J. Physiol. 273(2 Pt 2):R725-30).Similarly, rats offered a varied and palatable diet which mimics theso-called Western diet of humans (cafeteria diet) become obese due tohyperphagia (Rogers & Blundell (1984) Neurosci. Biobehav. Rev.8(4):441-53). Likewise, animals exposed to high-fat (HF) diets developobesity and exhibit reductions in insulin and leptin sensitivity (Clegg,et al. (2011) Physiol. Behav. 103(1):10-6; Hariri & Thibault (2010)Nutr. Res. Rev. 23(2):270-99) Such models are useful for demonstratingin vivo response for compounds of the present invention and exploringkey concepts such as dose-response.

Mouse Models of Diet-Induced Obesity.

Diet-induced obese (DIO) mice are the standard to study lipotoxicity invivo (Kennedy, et al. (2010) Disease Models & Mechanisms 3(3-4):156-66).High fat fed mice develop abnormalities in both the liver and pancreas.Depending on the genetic background, they develop insulin resistancewith or without β-cell atrophy and overt diabetes when on a high-fatdiet (Leiter & Reifsnyder (2004) Diabetes 53 Suppl. 1:S4-11; Tschop &Heiman (2001) Exp. Clin. Endocrinol. Diabetes 109(6):307-19). Strains ofmice that differ in propensity to develop β-cell atrophy include, e.g.,NONcNZOl0/LtJ (The Jackson Laboratory, Bar Harbor, Me.) that developsβ-cell atrophy and C57BL/6J (The Jackson Laboratory, Bar Harbor, Me.)that does not exhibit β-cell loss. Using these models, the effect ofnormal vs. high fat diet±test compound can be analyzed. Approximatelyhalf of NONcNZ0l10/LtJ males become diabetic and often develop isletatrophy on a high fat diet (Leiter (2009) Methods Mol. Biol. 560:1-17).Other strains that may be studied include the DIO mouse on the C57B1/6background which is not highly prone to β-cell loss but is a good modelof pre-T2D and obesity with elevated blood glucose and impaired glucosetolerance (Leiter (2009) Methods Mol. Biol. 560:1-17). C57B1/6KsJ db/dbmice develop diabetes associated with β-cell failure (Hummel, et al.(1972) Biochem. Genet. 7(1):1-13), which has been shown to becorrectable by MafA overexpression (Matsuoka, et al. (2015) J. Biol.Chem. 290:7647-7657), suggesting their use in an efficacy trial. Suchmodels are useful for demonstrating in vivo response for compounds ofthe present invention and exploring key concepts such as dose-response.

Animal Model of Metabolic Syndrome. New Zealand obese (NZO) mouse areobese and have severe T2DM. A number of genetic susceptibility loci thatfavor the development of adiposity and hyperglycemia have beenidentified in NZO mice. In addition to the leptin receptor gene, severalgenes of transcription factors were identified as potential candidategenes and orthologs of some of these genes have been linked to the humanmetabolic syndrome (Joost (2010) Results Probl. Cell Differ. 52():1-11). Such models are useful for demonstrating in vivo response forcompounds of the present invention and exploring key concepts such asdose-response.

Counter Screens.

Counter screens are often used to select among a library of compounds inorder to avoid off target effects. In the present invention, theactivity of compounds as modulators of HFN4α activity is the desiredtarget even though other off target effects may occur. Drugs that havebeen marketed for use in humans based on target effects other than HFN4αhave subsequently been shown to have activity as HNF4α activators(Alverine and Benfluorex; Lee, et al. (2013) ACS Chem. Biol.8(8):1730-6). Alverine has been marketed as a smooth muscle relaxant forgastrointestinal disorders, while Benfluorex was marketed as ananorectic agent. Benfluorex was known to be metabolized by cleavage ofan ester moiety into fenfluramine, a potent agonist of serotonin5-hydroxytryptamine 2(5-HT₂) receptors, an effect that was thought to berelated to its activity as an anorectic agent (Porter, et al. (1999) Br.J. Pharmacol. 128(1):13-20). However, modulation of 5-HT₂ receptors byBenfluorex was linked to undesirable cardiopulmonary side effects.Accordingly, based on these experiences with synthetic compounds,compounds and extracts of the present invention will be tested for offtarget effects on 5-hydroxytryptamine receptor activation using, e.g. afluorometric imaging plate reader (FLIPR) assay, which allows rapiddetection of rises in intracellular calcium levels in cells expressing ahuman 5-HT2_(A), 5-HT_(2B) or 5-HT_(2c) receptor in CHO-K1 cells. See,e.g., Porter, et al. (1999) Br. J. Pharmacol. 128(1):13-20. Othercounter screens may be chosen based on initial studies where toxiceffects may be linked to other off target actions.

Formulations

A substantially pure compound or extract comprising a compound of thisinvention can be combined with a carrier and provided in any suitableform for consumption by or administration to a subject. In this respect,the compound or extract is added as an exogenous ingredient or additiveto the consumable. Suitable consumable forms include, but are notlimited to, a dietary supplement, food ingredient or additive, a medicalfood, nutraceutical or pharmaceutical composition.

A food ingredient or additive is an edible substance intended to result,directly or indirectly, in its becoming a component or otherwiseaffecting the characteristic of any food (including any substanceintended for use in producing, manufacturing, packing, processing,preparing, treating, packaging, transporting, or holding food). A foodproduct, in particular a functional food, is a food fortified orenriched during processing to include additional complementary nutrientsand/or beneficial ingredients. A food product according to thisinvention can, e.g., be in the form of butter, margarine, sweet orsavory spreads, condiment, biscuits, health bar, bread, cake, cereal,candy, confectionery, soup, milk, yogurt or a fermented milk product,cheese, juice-based and vegetable-based beverages, fermented beverages,shakes, flavored waters, tea, oil, or any other suitable food.

A dietary supplement is a product taken by mouth that contains acompound or extract of the invention and is intended to supplement thediet. A nutraceutical is a product derived from a food source thatprovides extra health benefits, in addition to the basic nutritionalvalue found in the food. A pharmaceutical composition is defined as anycomponent of a drug product intended to furnish pharmacological activityor other direct effect in the diagnosis, cure, mitigation, treatment, orprevention of disease, or to affect the structure or any function of thebody of humans or other animals. Dietary supplements, nutraceuticals andpharmaceutical compositions can be found in many forms such as tablets,coated tablets, pills, capsules, pellets, granules, softgels, gelcaps,liquids, powders, emulsions, suspensions, elixirs, syrup, and any otherform suitable for use.

The term “carrier” as used herein means a material, composition orvehicle, such as a liquid or solid filler, diluent, excipient,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ, or portion of the body. Each.carrier should be compatible with the other ingredients of theformulation and not injurious to the subject. Some examples of materialsthat can serve as carriers include: (1) sugars, such as lactose, glucoseand sucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose, cellulose acetate, and hydroxyl propyl methylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernontoxic compatible substances employed in conventional formulations.

For preparing solid compositions such as tablets or capsules, thecompound or extract is mixed with a carrier (e.g., conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums) andother diluents (e.g., water) to form a solid composition. This solidcomposition is then subdivided into unit dosage forms containing aneffective amount of the compound of the present invention. The tabletsor pills containing the compound or extract can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction.

In particular embodiments of this invention, a consumable compositionincludes the compound or extract, a carrier and a preservative to reduceor retard microbial growth. The preservative is added in amounts up toabout 5%, preferably from about 0.01% to 1% by weight of the film.Preferred preservatives include sodium benzoate, methyl parabens, propylparabens, sodium nitrite, sulphur dioxide, sodium sorbate and potassiumsorbate. Other suitable preservatives include, but are not limited to,salts of edetate, (also known as salts of ethylenediaminetetraaceticacid, or EDTA, such a disodium EDTA).

The liquid forms in which the compound or extract of the invention isincorporated for oral or parenteral administration include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils as well as elixirs and similarvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product forreconstitution with water or other suitable vehicles before use. Suchliquid preparations may be prepared by conventional means withacceptable additives such as suspending agents (e.g., sorbitol syrup,methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); preservatives (e.g., methyl or propylp-hydroxybenzoates or sorbic acid); and artificial or natural colorsand/or sweeteners.

Methods of preparing formulations or compositions of this inventioninclude the step of bringing into association a compound or extract ofthe present invention with the carrier and, optionally, one or moreaccessory and/or active ingredients. In general, the formulations areprepared by uniformly and intimately bringing into association acompound or extract of the present invention with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product. As such, the disclosed formulation may consist of, orconsist essentially of a compound or extract described herein incombination with a suitable carrier.

When a compound or extract of the present invention is administered aspharmaceuticals, nutraceuticals, or dietary supplements to humans andanimals, they can be given per se or as a composition containing, forexample, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient incombination with an acceptable carrier.

A consumable product may be consumed by a subject to provide less than100 mg of a compound disclosed herein per day. In certain embodiments,the consumable provides between 10 and 60 mg/day of a tyraminecontaining hydroxycinnamic acid amide. The effective amount can beestablished by methods known in the art studies and be dependent uponbioavailability, toxicity, etc.

While it is contemplated that individual tyramine containinghydroxycinnamic acid amides may be used in the consumables of thisinvention, it is further contemplated that two or more of the compoundsor extracts could be combined in any relative amounts to produce customcombinations of ingredients containing two or more tyramine containinghydroxycinnamic acid amides in desired ratios to enhance productefficacy, improve organoleptic properties or some other measure ofquality important to the ultimate use of the product.

Molecular Target

HNF4α (hepatocyte nuclear factor 4α) is a global nuclear transcriptionfactor, regulating expression of many genes involved in maintainingbalanced metabolism (homeostasis). Notably, HNF4α is expressed in boththe liver (hepatocytes) and pancreas (β-cells). The expression andtranscriptional activity of HNF4α is decreased in NAFLD and T2DM in bothhuman liver cells and human pancreatic β-cells. HNF4α is mutated inMODY1, an autosomal dominant monogenic form of diabetes, providing humangenetic evidence for a direct role in diabetes pathogenesis. HNF4α geneexpression is down-regulated in T2D. In addition, free fatty acids,which are elevated in overweight and obese individuals, inhibit HNF4αactivity. In view of the fact that HNF4α haplo insufficiency causesdiabetes and HNF4α is down-regulated in T2D, restoration of or anincrease in HNF4α activity to the normal wild-type state would providean overall health and therapeutic benefit.

HNF4α-knockout rodent models exhibit the fatty liver phenotype, as wellas reduced lipogenesis, reduced de-novo cholesterol synthesis, reducedvery-low-density lipoprotein (VLDL) secretion and high-densitylipoprotein (HDL) biogenesis, as well as increased insulin intolerance.In addition, knockout mice show enhanced uptake of FFAs and reduceddegradation via β-oxidation. This results in hypocholesterolemia, lowblood triglyceride levels, and hepatic steatosis. All of this representsa significant dysregulation of lipid metabolism resulting from HNF4αdeficiency (Yin, et al. (2011) Arterioscler. Thromb. Vase. Biol.31(2):328-336; Hayhurst, et al. (2001) Mol. Cell Biol. 21(4)1393-1403;Martinez-Jimenez (2010) Mol. Cell. Biol. 30(3):565-577). By comparison,increased expression of HNF4α in the liver may increase transcription ofgenes that promote hepatic FFA oxidation, ketogenesis, and very-lowdensity lipoprotein (VLDL) secretion, as a means to deal with excess FFAaccumulation (Martinez-Jimenez (2010) Mol. Cell. Biol. 30(3):565-577).Therefore, HNF4α provides a target for mitigating the adverse effects ofFFAs, which are characteristically elevated in NAFLD.

In T2DM, HNF4α is responsible for direct regulation of genes involved inglucose transport and glycolysis. Without HNF4α in β-cells, rodentsexhibit defective glucose-stimulated insulin secretion in(β-cells—meaning decreased insulin secretion (Gupta, et al. (2005) J.Clin. Invest. 115(4)1006-15). It has been observed that HNF4α geneexpression is downregulated in individuals with T2DM, likely due toexposure to chronically elevated FFAs. In particular, it has been shownthat free palmitic acid (a C16 saturated FA) impairs pancreatic β-cellfunction and viability and suppresses normal insulin production due toactions on HNF4α (Lee, et al. (2013) ACS Chem. Biol. 8(8):1730-1736).Therefore, HNF4α provides a target for ameliorating the symptoms ofT2DM.

Metabolic Disorders

The term “metabolic disorder” refers to a disorder or condition thatoccurs when the body is unable to properly metabolize carbohydrates,lipids, proteins, and/or nucleic acids. Accordingly, in the context ofthe present invention disorders relating to abnormality of metabolismare encompassed in the term “metabolic disorder.” The term metabolicdisorder includes, but is not limited to, insulin resistance,hyperglycemia, diabetes mellitus (in particular T2DM), obesity, glucoseintolerance, hypercholesterolemia, hyperlipoproteinemia, dyslipidemia,hyperinsulinemia, atherosclerotic disease, coronary artery disease,metabolic syndrome, hypertension, or a related disorder associated withabnormal plasma lipoprotein, triglycerides or a disorder related toglucose levels such as pancreatic beta cell regeneration.

T2DM refers to a chronic disease or condition, which occurs when thepancreas does not produce enough insulin, or when the body cannoteffectively use the insulin it produces. This leads to an increasedconcentration of glucose in the blood (hyperglycemia). Based on studiesthat have established a relationship between plasma glucoseconcentrations, measures of glycemic exposure, and risk of diabeticretinopathy, the following criteria have been adopted for the diagnosisof diabetes mellitus: fasting plasma glucose greater than or equal to126 mg/dL (7.0 mmol/L); plasma glucose greater than or equal to 200mg/dL (11.1 mmol/L) at 2 hours following ingestion of 75 g anhydrousglucose in an oral glucose tolerance test; or random plasma glucosegreater than 200 mg/dL (11.1 mmol/L) in a person with symptoms ofdiabetes. Other important definitions include: impaired glucosetolerance with a plasma glucose equal to or greater than 140 mg/dL (7.8mmol/L) but less than 200 mg/dL (11.1 mmol/L) at 2 hours in the oralglucose tolerance test; and impaired fasting glucose with a fastingplasma glucose (FPG) equal to or greater than 100 mg/dL (5.6 mmol/L) butless than 126 mg/dL. A compound or extract of the invention is said tomodulate metabolism by decreasing one or more of fasting plasma glucose,plasma glucose following ingestion of 75 g anhydrous glucose, or randomplasma glucose levels below those referenced herein. Another endpointthat can be monitored as part of assessment of metabolic activity isblood levels of HbA1c; HbA1c is a measure of average glucose levels inblood over the past two to three months. Levels of HbA1c are used asclinical indicators of risk of diabetes, where increased levels areindicative of an increased risk of T2DM. Thus, reduction in HbA1c canalso be used to support an indication of glycemic control.

Obesity is a chronic, relapsing health risk defined by excess body fat.Total body fat can be accurately measured using hydrodensitometry anddual-energy x-ray absorptiometry (DEXA). Because body mass index (BMI),expressed as kilograms of weight divided by height in meters squared issimple and inexpensive to calculate, and correlates strongly with totalbody fat in non-elderly adults, it is commonly used as a surrogate fortotal body fat. Obesity is defined by the National Institutes of Healthas having a BMI of 30 kg/m² or higher. The relationships between BMI andrisks for death and major comorbidities vary by age, sex, race, andsmoking status, but, in general, are lowest in individuals with BMIs of18.5 kg/m² to 24.9 kg/m² and increase in a curvilinear or linear mannerwith BMIs of 25 kg/m² to approximately 40 kg/m². A compound or extractof the invention is said to modulate metabolism by decreasing meanand/or categorical body weight. Mean body weight is defined as thedifference in mean percent loss of baseline body weight in the activeproduct-treated versus placebo-treated group. Categorical body weight isdefined as the proportion of subjects who lose at least 5 percent ofbaseline body weight in the active product-treated versusplacebo-treated group. Secondary efficacy endpoints can include, but arenot limited to, improvements in blood pressure and pulse, lipoproteinlipids, fasting glucose and insulin, HbA1c (in T2DM), waistcircumference, and quality of life.

NAFLD, or “fatty liver,” is a metabolic disease characterized byexcessive accumulation of fat in the liver. NAFLD is characterized bypredominantly macrovesicular steatosis and the presence of visiblesteatosis in >5% of hepatocytes is generally accepted as a workingdefinition of a fatty liver (Kleiner, et al. (2005) Hepatology41:1313-1321) Nonalcoholic steatohepatitis or NASH is the most extremeform of NAFLD and is considered as a major cause of cirrhosis of liverof unknown etiology. The minimal criteria for the diagnosis of NASHinclude the presence of liver cell >5% macrovesicular steatosis,inflammation and ballooning, typically with a predominantlycentrilobular (acinar zone 3) distribution in adults. Steatohepatitis isnot simply the presence of inflammation and steatosis but is a specifichistologic entity (Kleiner, et al. (2005) Hepatology 41(6):1313-21;Brunt, et al. (1999) Am. J. Gastroenterol. 94:2467-2474; Ludwig, et al.(1980) Mayo Clin. Proc. 55:434-438; Neuschwander-Tetri & Caldwell (2003)Hepatology 37:1202-1219). A compound or extract of the invention is saidto modulate metabolism by measurably reducing the accumulation of fat inthe liver thereby improving liver function.

The term metabolic syndrome represents a cluster of laboratory andclinical findings that serve as markers for increased risk for coronaryheart disease, stroke, peripheral vascular disease and/or T2DM. Riskfactors associated with metabolic syndrome include abdominal obesity(i.e., excessive fat tissue in and around the abdomen), atherogenicdyslipidemia including but not limited to high triglycerides, low HDLcholesterol and high LDL cholesterol, elevated blood pressure, insulinresistance or glucose intolerance, prothrombotic state (e.g., highfibrinogen or plasminogen activator inhibitor-1 in the blood), and/orproinflammatory state (e.g., elevated C-reactive protein in the blood).A compound or extract of the invention is said to modulate metabolism byimproving components of metabolic syndrome and ultimately shown toprevent the development T2DM and reduce cardiovascular morbidity andmortality.

Metabolism Modulation

This invention also provides methods for modulating metabolism toameliorate, prevent or treat a metabolic disorder. In accordance withsuch methods, an effective amount of a compound or extract of thisinvention is administered to a subject in need of treatment so that thesubject's metabolism is modulated thereby addressing the underlyingpathogenesis of one or more metabolic disorders and promoting thehealth, well-being, and quality of life of the subject. The term“subject” as used herein refers to an animal, preferably a mammal. Insome embodiments, the subject is a veterinary, companion, farm,laboratory or zoological animal. In other embodiments, the subject is ahuman.

A subject in need of treatment includes a subject with observablesymptoms of a metabolic disorder (e.g., a subject with abnormal glucoseor lipid metabolism), as well as a subject who has no disorder but hasbeen observable symptoms of a metabolic determined to be susceptible todeveloping the metabolic disorder (i.e., a subject at risk of developingthe metabolic disorder). For example, according to the American HeartAssociation, metabolic syndrome (which raises the risk of heart disease,diabetes, stroke, and other health problems) is diagnosed when any threeof the following five risk factors are present: high blood glucose(sugar); low levels of HDL (“good”) cholesterol in the blood; highlevels of triglycerides in the blood; large waist circumference or“apple-shaped” body; or high blood pressure.

By way of further illustration, autoantibodies to insulin (IAA);glutamic acid decarboxylase (GAD); and an islet cell member of thereceptor type of the tyrosine phosphate family termed IA-2 have beenidentified as markers that predate the clinical onset of T2DM. See,e.g., U.S. Pat. Nos. 6,391,651 and 6,316,209. Similarly, C-reactiveprotein (CRP), apolipoprotein CIII, and plasma homocysteine levels havebeen identified as markers for identifying subjects at risk for highcholesterol (or hypercholesterolemia or hyperlipidemia) See, e.g., US2004/0198656; Yeh (2004) Can. J. Cardiol. 20(Suppl B):93-96B; andGeisel, et al. (2003) Clin. Chem. Lab. Med. 41(11):1513-7. Additionalfactors that can be used, alone or in combination, to determine whethera subject is at risk or predisposed to developing hypercholesterolemiainclude, without limitation, heredity (i.e., familialhypercholesterolemia), high blood pressure, history of smoking, alcoholconsumption, diabetes, obesity, physical inactivity, age and sex (i.e.,post-menopausal women over the age of 50), and stress.

The term “effective amount” as used herein means an amount of thecompound, extract, or formulation containing the compound or extract,which is sufficient to significantly improve a disorder. Of concern whendetermining an effective amount to be used in humans is balancing thedesired effects (benefits) against risks associated with use of acompound. At issue for such risk/benefit assessments is the types ofadverse effects observed and the likelihood that they will occur. Alsoconsidered is the fact that the effective amount may vary with theparticular disorder being treated, e.g., diabetes mellitus or obesity,the age and physical condition of the end user, the severity of thecondition, the duration of the treatment, the particular carrierutilized, and like factors.

In general, a suitable daily dose of a compound or extract of theinvention will be that amount of a compound or extract which is thelowest dose that is effective at producing a desired benefit, in thiscase an effect that improves metabolism of fats and sugars andconsequently supports overall health and well-being. Such an effectivedose will generally depend upon the factors described herein. For oraladministration, the dose may range from about 0.0001 mg to about 10grams per kilogram of body weight per day, about 5 mg to about 5 gramsper kilogram of body weight per day, about 10 to about 2 grams perkilogram of body weight per day, or any other suitable dose. If desired,the effective daily dose of the compound or extract may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. Preferred dosing is one administration per day.

The compound or extract of the invention can be used alone or incombination with a particular diet (e.g., foods with a low glycemicindex) or standard of care.

Administration of a compound or extract of the invention modulates themetabolism of a subject thereby addressing the underlying pathogenesisof one or more metabolic disorders and/or promoting the health,wellbeing, and quality of life of the subject. Ideally, an effectiveamount of a compound or extract provides a measurable improvement in thelevels or activity of one or more metabolic compounds. Examples includeHNF4α activity, insulin-like growth factor levels (such as insulin-likegrowth factor 1 or IGF-1), blood sugar levels, insulin levels, C peptidelevels, triglyceride levels, free fatty acid levels, blood uric acidlevels, microalbuminuria levels, glucose transporter expression,adiponectin levels, total serum cholesterol levels, high densitylipoprotein (HDL) levels, and/or low density lipoprotein (LDL) levels.

More particularly, administration of a compound or extract of theinvention improves metabolism, liver function, fasting plasma glucoselevels, postprandial plasma glucose levels, glycosylated hemoglobinHbA1c, body weight, insulin sensitivity, serum lipid profile byimproving lipid clearance, or a combination thereof. In particularembodiments, use of a compound or extract of the invention preferablyprevents, slows the progression of, delays or treats a metabolicdisorder such as T2DM, impaired glucose tolerance, impaired fastingblood glucose, hyperglycemia, postprandial hyperglycemia,hyperinsulinemia, NASH, NAFLD, or metabolic syndrome; slows theprogression of, delays or treats pre-diabetes; improves glycemic controland/or reduces fasting plasma glucose, postprandial plasma glucoseand/or glycosylated hemoglobin HbA1c; prevents, slows, delays orreverses progression of impaired glucose tolerance, impaired fastingblood glucose, insulin resistance or metabolic syndrome to T2DM;prevents, slows the progression of, delays, prevents or treats acomplication of diabetes mellitus such as cataracts or a micro- ormacrovascular disease, such as nephropathy, retinopathy, neuropathy,tissue ischemia, diabetic foot, dyslipidemia, arteriosclerosis,myocardial infarction, acute coronary syndrome, unstable anginapectoris, stable angina pectoris, stroke, peripheral arterial occlusivedisease, cardiomyopathy, heart failure, heart rhythm disorders orvascular restenosis; reduces body weight and/or body fat, or prevents anincrease in body weight and/or body fat, or facilitates a reduction inbody weight and/or body fat; prevents, slows, delays or treats diseasesor conditions attributed to an abnormal accumulation of ectopic fat, inparticular liver fat; maintains and/or improves insulin sensitivityand/or treats or prevents hyperinsulinemia and/or insulin resistance;reduces fat deposits; prevents, slows, delays or reverses progression offatty liver to NASH; and/or prevents, slows, delays or reversesprogression of NASH to cirrhosis, end-stage liver disease and/orhepatocellular carcinoma.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Assessing Indicators of Metabolic Activity: Materials andMethods

Expression of Insulin and HNF4α.

RNA was purified using a RNEASY® chromatographic separation andisolation kits (Qiagen), and converted to cDNA using the gScript™ cDNASuperMix (Quanta Biosciences). Q-PCR was conducted with cDNAcorresponding to 2 μg of RNA using an Opticon Real-Time System (MJResearch) and QPCR SuperMix (BioPioneer). See All mRNA values werenormalized to 18S rRNA values and are expressed as fold changes overvehicle-treated control.

Counter-Screen for Estrogenic Activity.

Estrogenic activity was monitored by co-transfection of a reporterplasmid containing a multimerized E-box 5′ of a minimal promoter fusedto the Firefly luciferase gene (4RTK-luc) with wild-type E47 or E47MER(Kiselyuk, et al. (2010) J. Biomol. Screen 15(6):663-70). HeLa cellswere transfected using polyethylenimine, 0.2 μg 4RTK-Luc plasmid andeither 0.3 μg of human E47, E47MER or pMSCVhph vector in 50 μl ofserum-free Dulbecco's modification of Eagle medium per well.Transfections included Renilla luciferase (pRL-TK) plasmid as a controlfor transfection efficacy. Transfection conditions were as described inthe PPRE-Luc reporter assay of Kiselyuk, et al. ((2010) J. Biomol.Screen 15(6):663-70). Sixteen hours after transfection, culture mediawere changed and maintained for 48 hours with tamoxifen and/or compoundor vehicle (DMSO). Cells were then lysed and assayed for luciferaseactivity using the Promega DUAL-LUCIFERASE® reporter assay kit (PromegaCorp., Madison, Wis.), and luminescence was measured using the Veritas™Microplate Luminometer (Turner Biosystems, Sunnyvale, Calif.). Data werenormalized to Renilla luciferase (pRL-TK) and expressed as fold-changeover vehicle alone.

Inhibition of HNF4α GFP Expression. Using the insulin promoter assaydescribed herein, activity of HNF4α was assessed in the presence ofBI-6015 (0, 2.5, 5 μM), a known antagonist of HNF4α (Kiselyuk, et al.(2012) Chem. Biol. 19(7):806-818), in combination withN-trans-caffeoyltyramine (0, 5, 10, 20 μM).

Hepatic Microsome Assay.

Hepatic microsomes stability assays were performed in accordance withknown methods (Peddibhotla, et al. (2013) ACS Med. Chem. Lett.4:846-851). Briefly, 3 μL of 25 μM compound solution in acetonitrilewere incubated with 123 μL of mouse, human or rat liver microsomes(Xenotech, Kansas City, Kans.). After preincubation at 37° C. for 10minutes, enzyme reactions were initiated by adding 120 μL ofNADPH-generating system (2 mM NADP⁺, 10 mM glucose-6-phosphate, 0.4 U/mlglucose-6-phosphate dehydrogenase, and 5 mM MgCl₂) in the presence of100 mM potassium phosphate buffer (pH 7.4) The final concentration ofeach compound used was 1 μM. The microsomal concentrations used were 1.0mg/mL. Compounds were incubated in microsomes for 0, 5, 15, 30 and 60minutes. The reactions were stopped by the addition of ice cold ACN andthe reaction mixtures were centrifuged at 10,000 g for 10 minutes beforethe supernatant was removed for analysis. A 10 μL portion of theresulting extract was injected on a Thermo HPLC system equipped with PALCTC plate sampler (96-well plate), Dionex Ultimate 3000 binary pump(flow rate at 0.600 mL/min), Dionex Ultimate 3000 thermostatted columncompartment (temperature at 40° C.), Thermo Endura Mass Spectrometer(ESI source), using a Thermo Scientific Accucore C18 (2.6 μM, 2.1×50 mm)column. A gradient was run starting at 95% H₂O (0.1% formic acid) and 5%ACN (0.1% formic acid) during the first 0.5 min, then under gradientcondition of 5-100% ACN (0.1% formic acid) from minute 0.5 to 3.5,finishing at 95% H₂O (0.1% formic acid) and 5% ACN (0.1% formic acid)over 0.5 min, with another 1 min at 95:5 to re-equilibrate.

Lipid Clearance in HepG2 and T6PNE Cells (Steatosis Assay).

The steatosis assay was carried out as described (Kiselyuk, et al.(2012) Chem. Biol. 19(7):806-818) with the exception of the drugconcentration, which was 20 μM for N-trans-caffeoyltyramine with 0.25 mMpalmitate in HepG2 cell lines, and 10 μM of eitherN-trans-caffeoyltyramine, N-trans-caffeoyltyramine, orpcoumaroyltyramine, with 0.25 mM palmitate in T6PNE cell lines.Steatosis was assessed using the Oil Red O Method for Fats kits (PolyScientific; Warrington, Pa.), per manufacturer's guidelines. Briefly,frozen tissue slides or fixed cells were incubated in neat propyleneglycol for 2 minutes and Oil Red O solution for 15 hours for slides or 1hour for fixed cells, differentiated in 85% propylene glycol solutionfor 1 minute, washed twice with distilled water and stained inHematoxylin of 10 seconds. Slides were mounted with glycerin jellymounting medium.

Alkaline Phosphatase (ALP) Quantitation.

Increased levels of ALP in blood are considered indicative of liverfunction abnormalities. Thus, ALP was assayed in accordance with knownmethods (Kiselyuk, et al. (2012) Chem. Biol. 19(7):806-818). Briefly,prior to sacrifice, blood was drawn and analyzed using a Vet Scan bloodanalyzer, measuring alkaline phosphatase (ALP, IU/L), alanineaminotransferase (ALT, IU/L), gamma glutamyl transferase (GGT, IU/L),bile acids (BA, μmol/L), total bilirubin (TBIL, mg/dL), albumin (ALB,g/dL), blood urea nitrogen (BUN, mg/dL), and cholesterol (CHOL, mg/dL).

Triglyceride (TG) Quantitation.

TG quantity was assayed using a Triglyceride Colorimetric Assay Kit(Cayman Chemicals; Ann Arbor, Mich.) according to the manufacturer'sinstructions.

Lipid Droplet Size Analysis.

All slides were scanned at a magnification of 20× using the AperioScanscope FL system (Aperio Technologies Inc.; Vista, Calif.). Theappropriate dyes were assigned and illumination levels were calibratedusing a preset procedure; the parameters were saved and applied to allslides. The acquired digital images represented whole tissue sections.Sections were evaluated for image quality. All acquired images weresubsequently placed in dedicated project folders, and stored on adesignated local server. Selected areas of the slides were selectedusing Aperio Imagescope (version 12 Aperio Technologies Inc.). Foranalysis, slides were viewed, whole tissue areas were selected andanalyzed using the web-based Image Scope viewer. Slides were quantifiedusing the ‘Color Deconvolution v9’ algorithm for oil redo staining(version 11 Aperio Technologies Inc.). The algorithm was optimized usinga preset procedure to maximize the strong red color positive oildroplets signal to noise ratio and the subsequent macro was saved andapplied to all slides.

HNF4α Immunostaining in Organ Samples.

Samples were harvested from mice, fixed in 4% paraformaldehyde andembedded in paraffin or O.C.T. freezing media (Sakura Finetek; Torrance,Calif.) Slides of 5 μm thickness were washed four times with PBS andtreated with 0.3% Triton™ in PBS for 10 minutes. Antigen retrieval wascarried out with CitriSolv™ (Fisher Scientific; Waltham, Mass.) for 10minutes in sub-boiling temperature. After washing with PBS for 10minutes, slides were incubated in blocking solution with 5% normaldonkey serum (Jackson Immuno Research; West Grove, Pa.) for 60 minutesat room temperature. Cells were fixed in 4% paraformaldehyde for 15minutes on 4° C. and washed with PBS, treated with 0.3% Tri Ton™ in PBSfor 10 minutes and blocked as previously described for slide samples.

Primary Antibodies.

HNF4α antibodies were used (#sc-6556, Santa Cruz Biotechnology; SantaCruz, Calif. and #3113, Cell Signaling Technology; Danvers, Mass.). Forfluorescent imaging, samples were incubated with ALEXA FLOUR® 488green-fluorescent dye or Rhodamine labeled anti-mouse, rabbit or goatand nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole)Controls using secondary antibodies alone were used to ensurespecificity of immunostaining. Fluorescently labeled sections wereanalyzed with a conventional inverted microscope (Olympus, PlanFl40x/0.60) or with a confocal microscope equipped with a krypton/argonlaser.

Bioavailability Determinations.

Male C57BL/6 mice were administered N-trans-caffeoyltyramine orN-trans-feruloyltyramine via IV, intraperitoneal or oral route (threemice for each route) (Table 2).

TABLE 2 Route Formulation Dosage (mg/kg) IV 1 mg/mL in 75% PEG 300/25%2.0 water, clear solution Oral 3 mg/mL in 0.5% methyl 30.0 cellulose,homogenous opaque suspension with fine particles IP 3 mg/mL in 5%DMSO/5% 30 Polysorbate 89/90% water, clear solution

A blood sample from each mouse was drawn at 0.25, 0.5, 1, 2, 4, 6 and 24hours after administration. An 8 μL aliquot of blood was used foranalysis. After adding 200 μL of an internal standard comprising 100ng/mL Labetalol, 100 ng/mL dexamethasone, 100 ng/mL tolbutamide, 100ng/mL Verapamil, 100 ng/mL Glyburide, and 100 ng/mL Celecoxib in ACN,the mixture was vortex-mixed and centrifuged at 12000 rpm for 15 minutesat 4 QC to pellet precipitated protein. Four μL of the supernatant wasinjected for LC-MS/MS analysis. Bioavailability (%) was calculated usingAUC_(0-inf) (% AUC_(Extra)<20%) or AUC_(0-last) (% AUC_(Extra)>20%) withnominal dose.

pH Stability Assessment.

Individual stock solutions were prepared in DMSO at concentrations of 10mg/mL. Four different buffer solutions were prepared to achievesolutions with a pH of 2, 7.4, 8.5 and 10. For each pH assay, 5 μL ofstock solution was added to 245 μL of buffered solution to a 2 mL tube,vortexed and incubated in a 37° C. water bath. At each timepoint, 50 μLaliquots were taken, neutralized and analyzed via HPLC analysis using aDAD detector at 280 nm. The fold change of the peak area at 280 nm wasanalyzed for the initial and final timepoint, 0.5 and 72 hours,respectively.

Example 2: Assessing Compounds for Activity as HNF4α Agonists

Given the role of HNF4α in maintaining a heal thy metabolism in humans,test compounds were screened for activity as HNF4α agonists (eitherdirect or indirect effects). Using a known insulin promoter-reporterassay, Kiselyuk and colleagues (2010. J. Biomol. Screen 15(6):663-70),screened a library of compounds for activity to promote insulinactivation. They identified compound 1 as an insulin activator(Kiselyuk, et al. (2012) Chem. Biol. 19(7):806-18) and the compound wassubsequently shown to possess HNF4α agonistic activity in an ornithinetranscarbamoylase (OTC) promoter assay. The OTC promoter is known to beresponsive to HNF4α in transient transfection assays (Inoue, et al.(2002) J. Biol. Chem. 277:25257-65).

To identify plant compounds that have similar bioactivity as thissynthetic agent (compound 1), a bioinformatics approach was taken topredict, from the set of all known plant compounds, a targeted sub-setwith the desired HNF4α agonistic activity. Using a number of algorithmsin combination with training data (i.e., positive data), models werebuilt around important features of the positive data, which werepredictive of the desired biological activity. More specifically, a setof 18 synthetic compounds with known ability to affect HNF4α activity(e.g., compound 1) were included in the positive data set. Thesestructures were used to search a database of plant compounds forchemical structures that had similar structural features. A number ofmetrics were used to measure similarity based on concepts from thefields of graph theory and information theory, either solely or incombination.

Plant compounds in the top 10th percentile of similarity to the 18target structures were selected and compounds predicted to be potentialagonists of HNF4α activity given their chemical structural features werescreened in the HNF4α assay. The results of the screening identified aclass of plant tyramine containing hydroxycinnamic acid amides (i.e.,N-trans-caffeoyltyramine, N-cis-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyltyramine) that are able to actas HNF4α modulators. Notably, N-trans-caffeoyltyramine was determined tobe roughly an order-of-magnitude more potent than Alverine in activatingHNF4α (FIG. 1 ) Due to hydroxyl derivatization of both phenyl rings,N-trans-caffeoyltyramine is less lipophilic and therefore expected to bemore bioavailable. Overall, the increased potency and expected enhancedbioavailability indicated that N-trans-caffeotyramine and other tyraminecontaining hydroxycinnamic acid amides would be expected to be moredesirable compounds for use in the methods disclosed herein.

Secondary experiments were performed to demonstrate that these compoundsdirectly modulate HNF4α activity. In particular, it was demonstratedthat insulin (FIG. 2 ) and HNF4α (FIG. 3 ) gene expression wereupregulated (e.g., as determined by quantitative PCR analysis) in thepresence of N-trans-caffeoyltyramine and N-trans-feruloyltyramine. Inaddition, it was found that p-coumaroyltyramine also upregulated insulinand HNF4a gene expression; however, cis-feruloyltyramine,N-coumaroyldopamine, N-transferuloyloctopamine and p-coumaroyloctopaminewere inactive. Further, using the insulin promoter assay,N-trans-caffeoyltyramine-mediated increases in insulin expression wereinhibited by BI-6015, a known HNF4α antagonist (FIG. 4 ). In addition,it was shown that N-trans-caffeoyltyramine and N-trans-feruloyltyraminedid not exhibit estrogenic activity (FIG. 5 ).

Using human, rat and mouse hepatic microsomes, in vitro pharmacologyindicated that N-trans-caffeoyl tyramine was stable and that higherbioactivity in humans may be attributed to the longer half-life ofN-trans-caffeoyltyramine in human cells compared to mouse hepaticmicrosomes (Table 3). For human microsomes, the apparent majorbiotransformation pathway was oxidation of the left-hand aryl ring.

TABLE 3 Clearance Half-life Rate % Microsomes Amount* (minutes)(μl/min/mg) Remaining Mouse  1 μM 0.8 1762.2 0.4 10 μM 6.9 200.4 0.3 Rat 1 μM 2.1 674.2 0.6 10 μM 22.4 30.9 33.6 Human  1 μM 77.3 17.9 55.3 10μM 262.3 5.3 85.4 *Amount of N-trans-caffeoyltyramine

Analysis of HepG2 liver cells treated with N-trans-caffeoyltyramine (20μM) or N-trans-feruloyltyramine (20 μM) indicated that these compoundswere capable of clearing harmful fats from the liver, as evidenced byOil Red O staining for fats, and further inhibited accumulation of fatsin HepG2 liver cells treated with 0.25 mM palmitate. A similarinhibition of fat accumulation was observed in T6PNE cells treated with0.25 mM palmitate and 10 μM N-trans-caffeoyltyramine, 10 μMN-trans-feruloyltyramine or 10 μM p-coumaroyltyramine.N-trans-caffeoyltyramine reduced lipid accumulation when palmitate wasadded prior to compound administration (FIG. 6 ).

In addition to performing assays demonstrating beneficial effects of thecompounds of the present invention, initial safety/toxicity assays wereperformed. The collective results of these analyses are presented inTable 4.

TABLE 4 N-trans- N-trans- caffeoyl feruloyl p-coumaroyl Assay tyraminetyramine tyramine HNF4α Activity + + + HNF4α mRNA + + + InsulinmRNA + + + Estrogenic Counter-Screen + + + Fat Clearance + + ND pHStability Acid Stable Stable Acid Stable Bioavailability ~11% ~7% ND ND,not determined

Example 3: Efficacy in Diet-Induced Obese Mice

In addition to demonstrating in vitro efficacy of the compounds of thepresent invention, experiments were performed in vivo in animal modelsof human disease, i.e., diet-induced obese mice. The experiments wereperformed to establish feeding and treatment regimen, dosing andadministration regimens, as well as to provide evidence of beneficialeffects of N-trans-caffeoyltyramine on glucose and lipid homeostasis,hepatic steatosis, β-cell function and hepatocyte function. Twelve mice(10 weeks old) were fed a high-fat diet for four weeks to induceobesity. After four weeks, and while on the high-fat diet, six mice wereadministered 5% DMSO or 120 mg/kg N-trans-caffeoyltyramine twice a dayintraperitoneally for 14 days. One hour after the last i.p. injection ofDMSO or N-trans-caffeoyl tyramine, the animals were sacrificed and bloodand organ (liver, kidney, gut and pancreas) samples were collected.Organ samples were subjected to histological, RNA, triglyceride andprotein analyses. Notably, the mice in this study did not exhibit anytoxic effects at any of the doses tested. The mice receiving treatmentdisplayed levels of activity, alertness, grooming, and appetiteconsistent with the control group. None of the treated mice exhibitedweight loss, sickness, or abnormal behaviors compared to the controlgroup.

Results showed that N-trans-caffeoyltyramine treatment decreased lipidaccumulation and significantly increased HNF4α expression (P=0.0042) inthe liver, in particular nuclear expression of HNF4α (FIG. 7 ).Immunostaining results indicated that N-trans-caffeoyltyramine increasedHNF4α activity. In addition, lipid droplet sizes in the liver werereduced in N-trans-caffeoyltyramine-treated animals levels of alkalinephosphatase (FIG. 8 In addition, levels of alkaline phosphatase (FIG. 9) and triglycerides (FIG. 10 ) were significantly reduced in micetreated with N-trans-caffeoyltyramine. The reduction in liver fat anddroplet size, and decrease in alkaline phosphatase demonstrate thebeneficial effects of increasing HNF4α activity. Given that alkalinephosphatase and triglyceride levels are often a routine part of bloodtesting in humans, with elevated levels being an indication of poorliver functioning, obesity and metabolic syndrome, alkaline phosphataseand triglyceride levels would provide useful markers for assessing theeffects of the tyramine containing hydroxycinnamic acid amides in humansadministered compounds of the present invention. In the pancreas, HNF4αexpression was increased in N-trans-caffeoyltyramine-treated animals, ascompared to DMSO control mice (FIG. 11 ). Similarly, HNF4α expressionwas increased in intestines upon administration ofN-trans-caffeoyltyramine (FIG. 12 )

These in vivo data demonstrated a correlation between HNF4α expressionand liver fat levels. In addition, results showed thatN-trans-caffeoyltyramine increased HNF4α activity in vivo and producedbeneficial effects on lipid, triglyceride, alkaline phosphatase andHNF4α levels.

Example 4: Evaluation of Compound-Related Toxicity

Given the need to balance benefits and risks of the compounds of thepresent invention, in vivo toxicity studies in laboratory animals (e.g.,mice, rats, dogs) are typically performed. Such studies are typicallyperformed consistent with Good Laboratory Practice (GLP) regulations toensure reliability and reproducibility for regulatory purposes. Ifcompounds are to be administered for periods of weeks to months to yearsin humans, chronic toxicity studies typically are performed (studies offrom six months to one year in duration). For compounds to be used infoods, oral toxicity studies are recommended.

The purpose of chronic toxicity testing is to determine thetoxicological profile of a test compound. In the initial phase oftesting, a study will be performed in rats. A total of 160 SpragueDawley rats (80 males and 80 females) approximately 5-7 weeks old andweighing between 80-100 g each will be randomly selected and allocatedto treatment groups by weight; such that the mean body weights of eachgroup will not be significantly different. The test compound or extractwill be administered orally at dose levels of 0.5, 1 and 2 g/kg bodyweight per day to rats for a period of 180 consecutive days. The animalswill be observed daily for any clinical signs of toxicity (e.g. rbehavioral changes; skin and fur appearance; eating and drinking; etc.).At the end of the experiment, the animals will be subjected tohematological, biochemical and histopathological evaluation consistentwith standard toxicological methods.

Example 5: Isolation of Tyramine Containing Hydroxycinnamic Acid Amidesfrom Plant Sources

Ethanolic extracts were prepared from various plant species and planttissues thereof. Individual compounds were identified in the extracts byextracting dry plant powder material with 95% aqueous ethanol. Theethanol extract was concentrated and adsorbed onto celite and dry-loadedonto a C18 solid phase extraction column. The extract was desalted bywashing with two column volumes of water which were collected anddiscarded. Compounds were eluted with two column volumes of methanol andthe extract was concentrated to dryness. The extract was resuspended in1:1 Acetonitrile:water prior to analysis. Synthetic standards of knownconcentrations were used to generate calibration curves prior toanalysis. The listing of sources used in the analysis are presentedbelow in Table 5. Plants are displayed for each compound in descendingorder with the plants that produce the highest amount of compound on thetop of the list and the lowest producers at the bottom of the list.

TABLE 5 Genus species Plant Tissues (s) N-Trans-caffeoyltryamine Annonamuricate Seed, pulp, skin Annon spp. Seed, pulp, skin Tribulusterrestris Seed, fruit Cannabis sp. Seed, hull, leaf Annona cherimolaSeed, pulp, skin, leaf, wood Annona montana Leaf Solanum lycopersciumFruit Solanum tuberosum Tuber, peel Lycium barbarum Fruit, stemN-Trans-feruloyltyramine Annona sp. Seed, pulp, skin Annona cherimolaSeed, pulp, skin, leaf, wood Piper nigrum Fruit Tribulus terrestrisSeed, fruit Annona muricate Seed, pulp, skin Solanum lycopersicum FruitCannabis Seed, hull, leaf Capsicum frutescens Fruit Allium fistulosumAerial plant Solanum tuberosum Tuber, peel Zea mays Seed, stalk, leafAllium sativum Bulb Annona montana Leaf Annona squamosa Fruit Lyciumbarbarum Fruit, stem Capsicum annuum Fruit Ipomoea batatas PeelChenopodium quinoa Seed Armoracia rusticana Root Capsicum annuum Fruit,leaf, stem Fagopyrum esculentum Hull Eragrostis tef Seedp-Courmaroyltyramine Annona spp. Seed, pulp, skin Tribulus terrestrisSeed, fruit Solanum lycopersicum Fruit Annona muricate Seed, pulp, skinAnnona montana Leaf Annona cherimola Seed, pulp, skin, leaf, woodCannabis spp. Seed, hull, leaf Solanum tuberosum Tuber, peel Alliumfistulosm Aerial plant Zea mays Seed, stalk leaf Allium sativum BulbIpomoea batatas Peel

The amounts of N-trans-caffeoyltyramine, N-transferuloyltyramine andp-coumaroyltyramine present in certain ethanol extracts (% of extract,w/w) was determined. Quantification of the compounds was performed bynormalizing the results by the weight of the ethanol extracts. Theresults of these analyses are presented in FIG. 13 .

Example 6: Efficacy of Test Compounds in an Animal Model of NAFLD

Like the diet-induced obese mouse model, there are otherwell-established animal models for examining the benefits of compoundsin NAFLD.

Animals and Diets.

Adult male Sprague-Dawley rats (250-300 grams) will be obtained.Custom-prepared diets including control, High-fat only, and High-fatdiet containing the test compound or extract. Control diet will be alow-fat diet where 12% of total calories are from corn oil, while mostof the fat is linoleic acid. The High-fat (HF) diet will contain 60% oftotal calories as lard as well as 2% corn oil, and the diet will beenriched in oleic acid and the saturated fatty acids palmitic andstearic. Such a High-fat diet was previously used to induce NAFLD inrats (Carmiel-Haggai, et al. (2005) FASEB J. 19:136-138). Seven rats ineach of the 4 groups will be randomized and fed the diets for 4 weeks:Group I: control diet; Group II: HF diet, Group III: HF+0.5%compound/extract diet; Group IV: HF+1% compound/extract. The rats willbe placed on a 12-hour day/night cycle and provided ad libitum access tofood and water. At the end of 4 weeks, rats will be fasted 16-18 hours,anesthetized, and blood and liver samples will be collected forbiochemical and histological analyses.

Serum and Liver Triglyceride and Cholesterol.

Serum triglyceride and total cholesterol will be measured bycommercially available assay kits (e.g., Wako Diagnostics; Richmond,Va.). Total lipid will be extracted from liver samples (about 0.25 g)with chloroform-methanol mixture (2:1) and washed with 0.73% sodiumchloride solution. The organic and aqueous phases will be separated bycentrifugation at 2000 rpm for 10 minutes. The organic phase containingtotal lipid will be dried completely under nitrogen and lipid extractreconstituted in isopropanol. An aliquot of lipid extract will be usedto measure triglycerides and total cholesterol using assay kits (e.g.,from Wako Diagnostics).

Measurement of Serum and Liver Thiobarbituric Acid-Reactive Substances(TEARS).

Serum and liver TEARS will be measured as an index of lipid peroxidationproducts.

Liver Histology.

Liver samples will be fixed in 10% formalin and embedded in paraffin.Sections (5 μrn) will be stained with hematoxylin and eosin andevaluated by a pathologist who will be blinded from the experimentalgroups and conditions. Sections will be subjected to semi-quantitationfor assessing steatosis.

Statistical Analysis.

Data will be presented as mean+S.E. Statistical analyses for the groupswill be made using a two-tailed Student's t-test, and p<0.05 will beconsidered statistically significant.

Example 7: an Evaluation of the Safety and Efficacy of Test Compounds inTreating Nash in Subjects with T2DM

The objective of the study will be to assess whether the examplecompound or extract can improve liver health and liver fat content, ascompared with placebo, in subjects who suffer from T2DM and NASH. Thestudy also will include assessment of serum alanine aminotransferase(ALT) levels, and determining whether test compound or extract treatmentis more effective than placebo treatment in reducing liver fat contentwhen measured by MRI-derived proton density-fat fraction (MRI-POFF). Thecomparison of serum ALT levels and liver fat content between compound orextract treatment and placebo treatment will be conducted in adultsubjects with NASH and T2DM at week 24 (or the last postbaselineobservation).

The secondary objectives of the study will be to evaluate the effects ofthe test compound or extract compared with placebo treatment on liverheath by assessing serum AST levels after 24 weeks of treatment;evaluate the effects of treatment on glycosylated hemoglobin (HbA1c);evaluate the effects of treatment on liver fibrosis, as measured usingtransient Elastography with Fibroscan. Considered together, the resultswill allow for assessment of overall safety and tolerability of testcompound or extract treatment as compared with placebo treatment.

Additionally, several exploratory objectives will be included in thestudy design. For example, the effect of the test compound or extract onthe immune profile of subjects based on 1) a change from baseline inhigh-sensitivity C-reactive protein (hsCRP) and erythrocytesedimentation rate (ESR); 2) a change from baseline in serum levels oftumor necrosis factor alpha (TNF-α); 3) a change from baseline in levelsof transforming growth factor (TGF) beta; 4) a change from baseline inlevels of interleukin (IL) −2, −4, −6, −10, and −12; and interferon(IFN) gamma; and 5) a fluorescence-activated cell sorting (FACS)analysis, which will measure a change from baseline in immunologicalmarkers such as cluster of differentiation 3 (CD 3), CD4, CD8, CD25,CD40, CD56, CD69, CD127, forkhead box P3 (FOXP3+), IL17, and retinoicacid-related orphan receptor-γt (RORγt)). Yet another exploratoryobjective will be to evaluate the effects of the test compound orextract on blood inflammatory markers (TNF-α, fibroblast growth factor19 (FGF-19)), liver fibrosis or cell death markers (cytokeratin-18(CK-18), soluble Fas (sFas)), and oxidative stress markers such ashydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids(HODEs), oxoeicosatetraenoic acids (oxoETEs), oxooctadecadienoic acids(oxoODEs)) and ox-nonalcoholic steatohepatitis (ox-NASH). Moreover, thestudy will: evaluate the effect of the test compound or extract usingthe homeostatic model assessment of insulin resistance (HOMA IR) tomeasure insulin; evaluate the effect of the compound or extract on serumlipid profile (triglycerides, high-density lipoprotein (HDL),low-density lipoprotein (LDL), and total cholesterol); and evaluate theeffect of the compound or extract on GLP1 and adiponectin.

Safety or tolerability endpoints will be evaluated after 24 weeks oftreatment with the test compound or extract. Endpoints will includeassessment of: the number and severity of any reported adverse events;physical examination findings, clinical laboratory evaluations (serumchemistry, hematology, and urinalysis) and 12-lead electrocardiograms(ECGs) from baseline to study completion; and the number of subjectsthat withdraw from the study before completion of the protocol. Thesafety laboratory test results will be collected and measured at thefollowing time points during the study: days-1 and 3 and weeks 1, 2, 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or early withdrawal).

A total of 80 T2DM and NASH subjects will be randomized into two groups:one group will receive a placebo, once daily (n=40); and one group willreceive a dose of the test compound or extract, 80 mg, once daily(n=40). Although test compound or extract will be administered at a doseof 80 mg per day, the dose may be titrated based on subjecttolerability, or it may be set at a fixed amount for the duration of thestudy, regardless of tolerability.

Example 8: an Evaluation of the Safety and Efficacy of Test Compounds inTreating Nash in Obese Subjects

A study will be conducted according to the methods of Example 7, whereinthe only difference is that the subject inclusion criteria include therequirement that the subjects are obese, as defined as having a BMI of≥30 instead of T2DM.

Example 9: an Evaluation of the Safety and Efficacy of Test Compounds inTreating NAFLD in Subjects with T2DM

The purpose of this study will be to determine whether the test compoundor extract can improve liver fat content and liver health, as comparedwith placebo, in subjects who suffer from both T2DM and NAFLD byassessing magnetic resonance imaging-derived proton density fat fraction(MRI-PDFF) after 24 weeks of treatment.

The secondary objectives of this study will be: 1) to evaluate theeffects of test compound or extract treatment, as compared with placebotreatment, on liver health by assessing serum ALT levels after 24 weeksof treatment; 2) to evaluate the effects of test compound or extracttreatment, as compared with placebo treatment, on liver heath byassessing serum AST levels after 24 weeks of treatment; 3) to evaluatethe effects of test compound or extract treatment on glycosylatedhemoglobin (HbA1c); 4) to evaluate the effects of test compound orextract treatment on liver fibrosis, as measured using transientElastography with Fibroscan; and 5) to evaluate the overall safety andtolerability of test compound or extract treatment as compared withplacebo treatment. Exploratory objectives of this study include thoselisted in Example 7.

A total of 80 T2DM and NAFLD subjects will be randomized into twogroups: one group will receive a placebo, once daily (n=40) and onegroup will receive a dose of test compound or extract, 80 mg, once daily(n=40) as described in Example 7. Subjects will be screened at visit 1between days −28 and −2. At screening, subjects will undergo screeningprocedures meant to ensure that inclusion/exclusion criteria are met,including an abdominal MRI to quantitatively measure liver fat content.Subjects who meet inclusion/exclusion criteria based on the results ofscreening assessments will return to the study center on day −1 toundergo baseline assessments (visit 2). At the baseline visit,confirmation of inclusion/exclusion criteria will be performed, andassessments of baseline laboratory values, physical examinationfindings, and ECG results also will be performed.

Subjects will be required to have a certified histology report whichdocuments and assesses the degree of steatosis, lobular inflammation,hepatocyte ballooning, and fibrosis that confirms a diagnosis of NAFLD.

At visit 18 on week 24 (or at early termination), all subjects willundergo end of treatment assessments, including liver fat contentimaging by MRI and clinical laboratory safety assessments.

What is claimed is:
 1. An oral composition comprising: a compound fororal consumption having the structure of Formula I, or an isomer, salt,homodimer, heterodimer or conjugate thereof:

wherein R¹ is present or absent, and when present is a substituent onone or more ring atoms and is for each ring atom independently a hydroxygroup, halo group, substituted or unsubstituted lower alkyl group, orsubstituted or unsubstituted lower alkoxy group; the dashed bond ispresent or absent; N-trans-feruloyltyramine, or a salt thereof; and acarrier, wherein the compound for consumption having the structure ofFormula I is N-trans-caffeoyltyramine and comprises between 0.1% to 99%of the composition, wherein the oral composition is a powder.
 2. Thecomposition of claim 1, further comprising p-coumaroyltyramine.
 3. Thecomposition of claim 1, wherein the carrier is selected from at leastone of a sugar, a starch, cellulose, powdered tragacanth, malt, gelatin,talc, excipient, oil, glycol, polyol, ester, agar, buffering agent,alginic acid, isotonic saline, ethyl alcohol, pH buffered solutions, orpolyesters.
 4. The composition of claim 1, wherein the composition isformulated in a tablet or capsule.
 5. A dietary supplement, foodingredient or additive, a medical food, nutraceutical, or pharmaceuticalcomprising a compound for oral consumption having the structure ofFormula I, or an isomer, salt, homodimer, heterodimer or conjugatethereof:

wherein R¹ is present or absent, and when present is a substituent onone or more ring atoms and is for each ring atom independently a hydroxygroup, halo group, substituted or unsubstituted lower alkyl group, orsubstituted or unsubstituted lower alkoxy group; the dashed bond ispresent or absent; N-trans-feruloyltyramine, or a salt thereof; acarrier; and a preservative, wherein the compound for oral consumptionhaving the structure of Formula I is N-trans-caffeoyltyramine andcomprises between 0.1% to 99% of the dietary supplement, food ingredientor additive, medical food, nutraceutical, or pharmaceutical, wherein thedietary supplement, food ingredient or additive, a medical food, ornutraceutical is a powder.
 6. The dietary supplement, food ingredient oradditive, a medical food, nutraceutical, or pharmaceutical of claim 5,wherein the preservative is from about 0.01% to 1% by weight of thedietary supplement, food ingredient or additive, medical food,nutraceutical, or pharmaceutical.
 7. The dietary supplement, foodingredient or additive, a medical food, nutraceutical, or pharmaceuticalof claim 5, wherein the preservative is selected from at least one ofsodium benzoate, methyl paraben, propyl paraben, sodium nitrite, sulphurdioxide, sodium sorbate, potassium sorbate or salts of edetate.
 8. Theoral composition of claim 1, further comprising cinnamoyltyramine,sinapoyltyramine, 5-hydroyferultyramine, or a combination thereof. 9.The oral composition of claim 1, wherein the oral composition has a pHbetween 2 and 7.4.
 10. The oral composition of claim 3, wherein thecellulose is carboxymethylcellulose.
 11. The oral composition of claim1, wherein the N-trans-caffeoyltyramine comprises between 10% to 99% ofthe composition.
 12. The oral composition of claim 1, wherein thecomposition comprises about 10 mg to about 60 mg ofN-trans-caffeoyltyramine and N-trans-feruloyltyramine.
 13. The oralcomposition of claim 1, wherein the N-trans-caffeoyltyramine and theN-trans-feruloyltyramine are an extract of Cannabis (hemp) seed hull.14. The dietary supplement of claim 5, further comprisingcinnamoyltyramine, sinapoyltyramine, 5-hydroyferultyramine, or acombination thereof.
 15. The dietary supplement of claim 5, wherein thepH of the dietary supplement, food ingredient or additive, a medicalfood, or nutraceutical is between 2 and 7.4 has a pH between 2 and 7.4.16. The dietary supplement of claim 5, wherein the carrier is selectedfrom the group consisting of cellulose, wax, and ester.
 17. The dietarysupplement of claim 16, wherein the cellulose is carboxymethylcellulose.18. The dietary supplement of claim 5, wherein theN-trans-caffeoyltyramine comprises between 10% to 99% of the dietarysupplement, food ingredient or additive, a medical food, nutraceutical,or pharmaceutical.
 19. An oral composition comprising: a compound fororal consumption having the structure of Formula I, or an isomer, salt,homodimer, heterodimer or conjugate thereof:

wherein R¹ is present or absent, and when present is a substituent onone or more ring atoms and is for each ring atom independently a hydroxygroup, halo group, substituted or unsubstituted lower alkyl group, orsubstituted or unsubstituted lower alkoxy group; the dashed bond ispresent or absent; p-coumaroyltyramine, or a salt thereof; and acarrier, wherein the compound for oral consumption having the structureof Formula I is N-trans-caffeoyltyramine and comprises between 0.1% to99% of the composition, wherein the composition is a powder.
 20. Thecomposition of claim 19, wherein the carrier is selected from at leastone of a sugar, a starch, cellulose, powdered tragacanth, malt, gelatin,talc, excipient, oil, glycol, polyol, ester, agar, buffering agent,alginic acid, isotonic saline, ethyl alcohol, pH buffered solutions, orpolyesters.
 21. The composition of claim 19, further comprisingcinnamoyltyramine, sinapoyltyramine, 5-hydroyferultyramine, or acombination thereof.
 22. The composition of claim 19, wherein thecomposition is formulated in a tablet or capsule.
 23. The oralcomposition of claim 20, wherein the cellulose iscarboxymethylcellulose.
 24. The oral composition of claim 19, whereinN-trans-caffeoyltyramine comprises between 10% to 99% of thecomposition.
 25. A dietary supplement, food ingredient or additive, amedical food, nutraceutical, or pharmaceutical comprising a compound fororal consumption having the structure of Formula I, or an isomer, salt,homodimer, heterodimer or conjugate thereof:

wherein R¹ is present or absent, and when present is a substituent onone or more ring atoms and is for each ring atom independently a hydroxygroup, halo group, substituted or unsubstituted lower alkyl group, orsubstituted or unsubstituted lower alkoxy group; the dashed bond ispresent or absent; and p-coumaroyltyramine, or a salt thereof; acarrier; and a preservative, wherein the compound for oral consumptionhaving the structure of Formula I is N-trans-caffeoyltyramine andcomprises between 0.1% to 99% of the dietary supplement, food ingredientor additive, a medical food, nutraceutical, or pharmaceutical, whereinthe dietary supplement, food ingredient or additive, a medical food, ornutraceutical is a powder.
 26. The dietary supplement, food ingredientor additive, a medical food, nutraceutical, or pharmaceutical of claim25, wherein the preservative is from about 0.01% to 1% by weight of thedietary supplement, food ingredient or additive, a medical food,nutraceutical, or pharmaceutical.
 27. The dietary supplement, foodingredient or additive, a medical food, nutraceutical, or pharmaceuticalof claim 25, wherein the N-trans-caffeoyltyramine comprises between 10%to 99% of the dietary supplement, food ingredient or additive, a medicalfood, nutraceutical, or pharmaceutical.
 28. The dietary supplement ofclaim 25, further comprising cinnamoyltyramine, sinapoyltyramine,5-hydroyferultyramine, or a combination thereof.
 29. The dietarysupplement of claim 25, wherein the carrier is selected from the groupconsisting of cellulose, waxes, oils and esters.
 30. The oralcomposition of claim 29, wherein the cellulose iscarboxymethylcellulose.